| Objective:To investigate whether Aurora kinase B（Aurk B）is associated with the pathogenesis of atherosclerosis and how it can affect and regulate the development of atherosclerosis.By constructing an atherosclerosis animal model,the influence of Aurk B on the pathogenesis of atherosclerosis was assessed,and the mechanism employed by Aurk B to regulate the pathogenesis of atherosclerosis was explored using various cellular models.Methods:1.To assess the effect of Aurk B on the pathogenesis of atherosclerosis（1）A mouse model of atherosclerosis was constructed to verify the association between Aurk B expression and atherosclerosis.8-week-old healthy male Apo E-/-（Apolipoprotein E KO）mice with C57BL/6J background was divided randomly into two groups:（1）Apo E-/-normal feeding group（AND）,fed with normal food;（2）Apo E-/-high-fat feeding group（HFD）,fed with high-fat food.Early atherosclerosis and advanced atherosclerosis models were constructed by 8-week and 16-week high fat diet.Blood was drawn from the orbit and used for serum lipid measurement to make sure the model was successfully established,and then liver and spleen tissue samples were collected.The relative expression of Aurk B m RNA in liver,spleen was detected by q-PCR technology.（2）Using 8-week-old Apo E-/-healthy male mice with C57BL/6J background,an atherosclerotic model was constructed,and all the mice was divided randomly into four groups:（1）Low-dose AZD1152 group（AL）,fed with high-fat diet and injected with AZD1152（25mg/kg,twice a week）;（2）High-dose AZD1152 group（AH）,fed with high-fat diet,and injected with AZD1152（25 mg/kg,3 times a week）;（3）Control group（AC）,fed with high-fat diet,and injected with solvent（the same amount of solvent as used in the AZD1152-treated group,3 times a week）;（4）Normal diet group（AN）,fed with normal diet.All the groups except AN group were fed with high-fat diet for either 8 weeks or 16 weeks,and weighed weekly for weight monitoring.AZD1152 was administered intraperitoneally for 4 weeks since week 4 or 12 of high-fat feeding.At the endpoint,mouse sera and multiple tissue samples were collected.Blood lipid level indicators such as TG/TC/LDL was measured;The aortas were stained with oil red O to assess the area of plaque in the aorta,and the formation and composition of plaque at the aortic root valve of the mouse heart were evaluated using Oil red O staining,HE staining and Masson staining.So that,the effect of inhibiting Aurk B on the progression of atherosclerosis was comprehensively studied.2.To explore the mechanism used by Aurk B to regulate the development of atherosclerosis（1）To explore the effect of Aurk B on liver lipid metabolism:1)TG/TC levels and expression levels of lipid metabolism-related genes were analyzed using the liver samples from mice after 16-week high-fat diet;2)By adding Palmitic acid/oleic acid（PA/OA）mixture,a high-lipid model based on Hep G2cells was constructed to detect the relative expression changes of m RNA and protein levels of Aurk B in the context of lipid accumulation,and Aurk B inhibitor AZD1152 was used to investigated the effect of Aurk B on lipid metabolism by assessing the lipid droplet deposition in hepatocytes.（2）To explore the effect of Aurk B on the formation of macrophage-derived foam cells:1)To study the effect of Aurk B inhibitors on hematopoiesis in vivo,the cells in the bone marrow and spleen were numerated and the composition of blood lineage was analyzed by flow cytometry,the results of which could tell whether Aurk B inhibitors affect the formation of foam cells in plaque by affecting myeloid cell production;2)The bone marrow cells of 8-week-old C57BL/6J male mice were induced by M-CSF to generate bone marrow-derived macrophages,and further induced by oxidized low-density lipoprotein（ox-LDL）was added to further construct a macrophage-derived foam cell model.The protein level changes of Aurk B in the process of foam cell production were evaluated,and Aurk B inhibitor AZD1152 was added to study the effect of inhibiting Aurk B on the accumulation of lipid droplets by oil red O staining.Thus,the correlation between Aurk B and foam cell production could be elucidated,and the effect of Aurk B inhibition on the formation of macrophage-derived foam cells could also be explored.Results:1.After 8-week and 16-week high-fat diets,the serum levels of TG/TC/LDL in high-fat fed mice increased significantly,and the expression levels of Aurk B m RNA in the liver and spleen of mice in the HFD group were significantly higher than those in the AND group;2.Compared with the AC group,the AL group and AH group had a slightly decreased body weight,but without a significant difference.Also,the kidney/body weight ratio between the AC group and the AH group was not significantly different.After 8-week and 16-week high-fat diet,the treatment of Aurk B inhibitor did not reduce serum TG/TC/LDL levels relative to the AC group.En face oil red staining of the aorta showed that high-dose Aurk B inhibitor treament reduced total plaque area in the aorta at the 16-week high-fat diet timepoint.Arterial root HE staining showed that high-dose Aurk B inhibitor significantly reduced the thickness of aortic root plaque;Oil red O staining showed that both high and low doses of Aurk B inhibitor treatment reduced lipid accumulation in the plaques at both timepoints;Masson staining showed that high-dose Aurk B inhibitor treatment increased collagen content in mouse plaques at both time point.Compared with high-dose Aurk B inhibitor,low-dose Aurk B inhibitor only reduced lipid accumulation in plaques and increased collagen content in plaques;3.At 16-week high-fat diet timepoint,high-dose inhibitor treatment reduced TG/TC levels and lipid accumulation remarkably in mouse livers,and downregulated the expression levels of genes such as Hmgcr;4.Compared with the control group,there was no significant change in the protein level of Aurk B in Hep G2 cells after 24-h and 48-h PA/OA treatment,and there was no significant change in the proportion of oil red stained area after treatment of AZD1152;5.High-dose Aurk B inhibitor treatment did not significantly reduce the number of cells and CD11b+cells in the bone marrow and spleen,and the proportion of CD11b+cell clusters in peripheral blood;6.Compared with the control group,the relative expression of Aurk B protein increased at 6h,12 h,24 h and 48 h during foam cell formation,but there were statistical differences only at24-h and 48-h timepoints.After the addition of Aurk B inhibitors,lipid droplet accumulation decreases significantly in the foam cells.Conclusion:1.High expression of Aurk B is associated with atherosclerosis;2.Aurk B inhibition can effectively ameliorate the progression of atherosclerosis;3.Aurk B regulates liver lipid metabolism,but may not work by regulating hepatocytes;4.Aurk B inhibitors may inhibit the development of atherosclerosis by affecting macrophage-derived foam cell production. |
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